Simon Blank

Added sensitivity of component-resolved diagnosis in hymenoptera venom-allergic patients with elevated serum tryptase and/or mastocytosis

Anaphylaxis caused by hymenoptera venom allergy is associated with elevation of baseline serum tryptase (sBT) and/or mastocytosis in about 5% of patients. Up to now, no information has become available on single venom allergen sIgE reactivity and the usefulness of component‐resolved approaches to diagnose this high‐risk patient group. To address the component‐resolved sIgE sensitization pattern and diagnostic sensitivity in hymenoptera venom‐allergic patients with elevated sBT levels and/or mastocytosis, a panel of yellow jacket and honeybee venom allergens was applied on a widely used IgE immunoassay platform.

Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics

Hymenoptera stings can cause severe anaphylaxis in untreated venom‐allergic patients. A correct diagnosis regarding the relevant species for immunotherapy is often hampered by clinically irrelevant cross‐reactivity. In vespid venom allergy, cross‐reactivity between venoms of different species can be a diagnostic challenge. To address immunological IgE cross‐reactivity on molecular level, seven recombinant antigens 5 of the most important Vespoidea groups were assessed by different diagnostic setups.

Decline of Ves v 5‐specific blocking capacity in wasp venom‐allergic patients after stopping allergen immunotherapy

While allergen‐specific immunotherapy (AIT) is very efficient in hymenoptera venom (HV)‐allergic patients, long‐term outcome after finishing AIT is not well investigated, especially regarding mechanisms that are suggested to contribute to allergen‐specific tolerance. Here, we analyse the Ves v 5‐inhibitory activity of sera from wasp venom‐allergic patients using the novel cell‐free enzyme‐linked immunosorbent facilitated antigen binding (ELIFAB) assay. Compared to pre‐AIT, sera from patients undergoing AIT displayed an increased ability to inhibit Ves v 5 binding by IgE antibodies. In contrast, this inhibitory activity was reduced in patients having finished AIT 5–12 years ago. Allergen‐blocking capacity correlated with serum concentrations of Ves v 5‐specific IgG4 which rose during AIT but almost reached pretreatment levels in patients who had stopped AIT more than 5 years ago. These data raise questions about how long allergen tolerance is maintained in AIT‐treated HV‐allergic patients and suggest that the ELIFAB assay might be an easy‐to‐use tool assessing long‐term tolerance in patients treated with HV‐AIT.

Component-resolved evaluation of the content of major allergens in therapeutic extracts for specific immunotherapy of honeybee venom allergy

ABSTRACT Allergen-specific immunotherapy is the only curative treatment of honeybee venom (HBV) allergy, which is able to protect against further anaphylactic sting reactions. Recent analyses on a molecular level have demonstrated that HBV represents a complex allergen source that contains more relevant major allergens than formerly anticipated. Moreover, allergic patients show very diverse sensitization profiles with the different allergens. HBV-specific immunotherapy is conducted with HBV extracts which are derived from pure venom. The allergen content of these therapeutic extracts might differ due to natural variations of the source material or different down-stream processing strategies of the manufacturers. Since variations of the allergen content of therapeutic HBV extracts might be associated with therapeutic failure, we adressed the component-resolved allergen composition of different therapeutic grade HBV extracts which are approved for immunotherapy in numerous countries. The extracts were analyzed for their content of the major allergens Api m 1, Api m 2, Api m 3, Api m 5 and Api m 10. Using allergen-specific antibodies we were able to demonstrate the underrepresentation of relevant major allergens such as Api m 3, Api m 5 and Api m 10 in particular therapeutic extracts. Taken together, standardization of therapeutic extracts by determination of the total allergenic potency might imply the intrinsic pitfall of losing information about particular major allergens. Moreover, the variable allergen composition of different therapeutic HBV extracts might have an impact on therapy outcome and the clinical management of HBV-allergic patients with specific IgE to particular allergens.

Allergen-specific immunotherapy of Hymenoptera venom allergy – also a matter of diagnosis

ABSTRACT Stings of hymenoptera can induce IgE-mediated hypersensitivity reactions in venom-allergic patients, ranging from local up to severe systemic reactions and even fatal anaphylaxis. Allergic patients quality of life can be mainly improved by altering their immune response to tolerate the venoms by injecting increasing venom doses over years. This venom-specific immunotherapy is highly effective and well tolerated. However, component-resolved information about the venoms has increased in the last years. This knowledge is not only able to improve diagnostics as basis for an accurate therapy, but was additionally used to create tools which enable the analysis of therapeutic venom extracts on a molecular level. Therefore, during the last decade the detailed knowledge of the allergen composition of hymenoptera venoms has substantially improved diagnosis and therapy of venom allergy. This review focuses on state of the art diagnostic and therapeutic options as well as on novel directions trying to improve therapy.

The high molecular weight dipeptidyl peptidase IV Pol d 3 is a major allergen of Polistes dominula venom

Hymenoptera venom allergy can cause severe anaphylaxis in untreated patients. Polistes dominula is an important elicitor of venom allergy in Southern Europe as well as in the United States. Due to its increased spreading to more moderate climate zones, Polistes venom allergy is likely to gain importance also in these areas. So far, only few allergens of Polistes dominula venom were identified as basis for component-resolved diagnostics. Therefore, this study aimed to broaden the available panel of important Polistes venom allergens. The 100u2009kDa allergen Pol d 3 was identified by mass spectrometry and found to be a dipeptidyl peptidase IV. Recombinantly produced Pol d 3 exhibited sIgE-reactivity with approximately 66% of Polistes venom-sensitized patients. Moreover, its clinical relevance was supported by the potent activation of basophils from allergic patients. Cross-reactivity with the dipeptidyl peptidases IV from honeybee and yellow jacket venom suggests the presence of exclusive as well as conserved IgE epitopes. The obtained data suggest a pivotal role of Pol d 3 as sensitizing component of Polistes venom, thus supporting its status as a major allergen of clinical relevance. Therefore, Pol d 3 might become a key element for proper diagnosis of Polistes venom allergy.

Improved efficacy of allergen-specific immunotherapy by JAK inhibition in a murine model of allergic asthma

Background Allergen-specific immunotherapy (AIT) is the only curative treatment for type-1 allergies, but sometimes shows limited therapeutic response as well as local and systemic side effects. Limited control of local inflammation and patient symptoms hampers its widespread use in severe allergic asthma. Objective Our aim was to evaluate whether AIT is more effective in suppression of local inflammation if performed under the umbrella of short-term non-specific immunomodulation using a small molecule inhibitor of JAK pathways. Methods In C57BL/6J mice, a model of ovalbumin (OVA)-induced allergic airway inflammation and allergen-specific immunotherapy was combined with the administration of Tofacitinib (TOFA, a FDA-approved JAK inhibitor) from 48 hours prior to 48 hours after therapeutic OVA-injection. The effect of TOFA on human FOXP3+CD4+ T cells was studied in vitro. Results AIT combined with short-term TOFA administration was significantly more effective in suppressing total cell and eosinophil infiltration into the lung, local cytokine production including IL-1β and CXCL1 and showed a trend for the reduction of IL-4, IL-13, TNF-α and IL-6 compared to AIT alone. Furthermore, TOFA co-administration significantly reduced systemic IL-6, IL-1β and OVA-specific IgE levels and induced IgG1 to the same extent as AIT alone. Additionally, TOFA enhanced the induction of human FOXP3+CD4+ T cells. Conclusions This proof of concept study shows that JAK inhibition did not inhibit tolerance induction, but improved experimental AIT at the level of local inflammation. The improved control of local inflammation might extend the use of AIT in more severe conditions such as polyallergy, asthma and high-risk patients suffering from mastocytosis or anaphylaxis.

Component-resolved diagnostics to direct in venom immunotherapy: important steps towards precision medicine

Stings of Hymenoptera can induce IgE‐mediated systemic and even fatal allergic reactions. Venom‐specific immunotherapy (VIT) is the only disease‐modifying and curative treatment of venom allergy. However, choosing the correct venom for VIT represents a necessary prerequisite for efficient protection against further anaphylactic sting reactions after VIT. In the past, therapeutic decisions based on the measurement of specific IgE (sIgE) levels to whole venom extracts were not always straightforward, especially when the patient was not able to identify the culprit insect. In the last years, the increasing knowledge about the molecular structure and relevance of important venom allergens and their availability as recombinant allergens, devoid of cross‐reactive carbohydrate determinants, resulted in the development of an advanced component‐resolved diagnostics (CRD) approach in venom allergy. Already to date, CRD has increased the sensitivity of sIgE detection and enabled the discrimination between primary sensitization and cross‐reactivity, particularly in patients with sensitization to both honeybee and vespid venom. Hence, CRD in many patients improves the selection of the appropriate immunotherapeutic intervention. Moreover, the detailed knowledge about sensitization profiles on a molecular level might open new options to identify patients who are at increased risk of side‐effects or not to respond to immunotherapy. Therefore, increasing potential of CRD becomes evident, to direct therapeutic decisions in a personalized and patient‐tailored manner. Reviewed here are the state of the art options, recent developments and future perspectives of CRD of Hymenoptera venom allergy.

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential

&NA; Honeybee (Apis mellifera) venom (HBV) represents an ideal model to study the role of particular venom components in allergic reactions in sensitized individuals as well as in the eusociality of Hymenoptera species. The aim of this study was to further characterize the HBV components C1q‐like protein (C1q) and PDGF/VEGF‐like factor 1 (PVF1). C1q and PVF1 were produced as recombinant proteins in insect cells. Their allergenic properties were examined by determining the level of specific IgE antibodies in the sera of HBV‐allergic patients (n = 26) as well as by their capacity to activate patients basophils (n = 11). Moreover, the transcript heterogeneity of PVF1 was analyzed. It could be demonstrated that at least three PVF1 variants are present in the venom gland, which all result from alternative splicing of one transcript. Additionally, recombinant C1q and PVF1 from Spodoptera frugiperda insect cells exhibited specific IgE reactivity with approximately 38.5% of sera of HBV‐allergic patients. Interestingly, both proteins were unable to activate basophils of the patients, questioning their role in the context of clinically relevant sensitization. Recombinant C1q and PVF1 can build the basis for a deeper understanding of the molecular mechanisms of Hymenoptera venoms. Moreover, the conflicting results between IgE sensitization and lack of basophil activation, might in the future contribute to the identification of factors that determine the allergenic potential of proteins. HighlightsC1q‐like protein and PVF1 are honeybee venom components that induce sIgE in honeybee‐venom allergic patients.At least three PVF1 variants are present in the venom gland, resulting from alternative splicing of one transcript.Both proteins are unable to activate basophils, questioning their clinical relevance.The recombinant proteins can contribute to a deeper understanding of the molecular mechanisms of Hymenoptera venoms.

The basophil activation test differentiates between patients with alpha-gal syndrome and asymptomatic alpha-gal sensitization

Background: Galactose‐alpha‐1,3‐galactose (alpha‐gal) syndrome is characterized by the presence of serum specific IgE antibodies to alpha‐gal and delayed type I allergic reactions to the carbohydrate alpha‐gal after consumption of mammalian (red) meat products and drugs of mammalian origin. Diagnostics currently rely on patient history, skin tests, determination of serum specific IgE antibodies, and oral food or drug challenges. Objective: We sought to assess the utility of different basophil parameters (basophil reactivity and sensitivity, the ratio of the percentage of CD63+ basophils induced by the alpha‐gal–containing allergen to the percentage of CD63+ basophils after stimulation with anti‐Fc&egr;RI antibody [%CD63+/anti‐Fc&egr;RI], and area under the dose‐response curve [AUC]) as biomarkers for the clinical outcome of patients with alpha‐gal syndrome compared with subjects with asymptomatic alpha‐gal sensitization. Methods: In addition to routine diagnostics, a basophil activation test (Flow CAST) with different concentrations of alpha‐gal–containing allergens (eg, commercially available alpha‐gal–carrying proteins and pork kidney extracts) was performed in 21 patients with alpha‐gal syndrome, 12 alpha‐gal–sensitized subjects, and 18 control subjects. Results: Alpha‐gal–containing allergens induced strong basophil activation in a dose‐dependent manner in patients. Basophil reactivity at distinct allergen concentrations, the %CD63+/anti‐Fc&egr;RI ratio across most allergen concentrations, the AUC of dose‐response curves, and basophil allergen threshold sensitivity (CD‐sens) with pork kidney extract were significantly higher in patients with alpha‐gal syndrome compared with those in sensitized subjects. All parameters were negative in control subjects. Conclusion: The basophil activation test should be considered as an additional diagnostic test before performing time‐consuming and potentially risky oral provocation tests. The %CD63+/anti‐Fc&egr;RI ratio for all allergens and AUCs for pork kidney were the best parameters for distinguishing patients with alpha‐gal syndrome from subjects with asymptomatic alpha‐gal sensitization. GRAPHICAL ABSTRACT Figure. No caption available.